Mutant Ubiquitin Research Articles

Mutant ubiquitin and p62 immunoreactivity in cases of combined multiple system atrophy and Alzheimer’s disease

Recent studies have shown the co-existence of alpha-synuclein and phosphorylated tau (pTau) in several neurodegenerative diseases. Here, we report two autopsy cases of combined multiple system atrophy (MSA) and Alzheimer's disease (AD). In both cases, abundant alpha-synuclein-positive glial and neuronal cytoplasmic inclusions were found in the brainstem, amygdala and hippocampal formation. pTau-positive neurofibrillary tangles (NFTs) were widely distributed in case 1 (Braak stage VI) and moderate in case 2 (Braak stage III). Although alpha-synuclein and pTau pathology co-occurred in the hippocampus and entorhinal cortex, only a few neurons showed co-existence of these two proteins. Immunoreactivity for p62, a ubiquitin proteasome system related protein, was found in the majority of NFTs, but in only a small proportion of neuronal alpha-synuclein inclusions. In addition, UBB+1, a mutant form of ubiquitin and a marker for proteasomal dysfunction, was present in the majority of NFTs, whereas co-existence of alpha-synuclein and UBB+1 was found in only a few neurons. These findings indicate that alpha-synuclein and phosphorylated tau co-occur in certain brain regions in cases of combined MSA and AD and that the proteasomal pathways differ between alpha-synuclein- and pTau-bearing neurons.

Characterisation of the nascent polypeptide-associated complex in fission yeast

The nascent polypeptide-associated complex (NAC) is an abundant and phylogenetically conserved protein complex. It is composed of two subunits and interacts with nascent polypeptide chains emerging from the ribosome. It has been proposed to protect the nascent chains from premature interaction with other cell proteins, but has also been found to associate with DNA junctions, and to be involved in other processes including transcription regulation and mitochondrial protein import.Here, we characterize NAC in fission yeast. We find that NAC is associated with ribosomes, while a significant fraction remains in a free form. The NAC alpha subunit contains a ubiquitin-associated (UBA) domain, which is found in several proteins involved in the ubiquitin-proteasome pathway for protein degradation. However, NAC does not associate with ubiquitin chains and mutants lacking NAC did not exhibit any obvious defects in protein degradation. Accordingly, we find that the NAC UBA domain belongs to an ancient and distinct subgroup of the UBA family. In contrast to the situation with budding yeast, fission yeast cells devoid of NAC were not temperature sensitive. However, they displayed resistance to the amino acid analogue canavanine, in accordance with the idea that NAC is involved in protein quality control.

Modulation of HIV-1 Rev protein abundance and activity by polyubiquitination with unconventional Lys-33 branching

The HIV-1 Rev protein plays a key role in virus replication by allowing export to the cytoplasm of unspliced or singly-spliced RNAs. In this report, we investigated whether Rev is modified by ubiquitination or sumoylation. Whereas no evidence of sumoylation was obtained, transient expression experiments showed that ubiquitin conjugates to Rev as high molecular weight polyubiquitin chains. Mutation of the three lysine residues of Rev showed that the site of ubiquitin conjugation is Lys-115. Experiments with ubiquitin mutants including a single lysine at every seven possible position indicated that branching of the polyubiquitin chains mainly involves Lys-33. Mutation of Rev Lys-115 to arginine reduces markedly the steady state amount of the protein, but does not impair its ability to export RNA via the Rev response element. These observations support the notion that polyubiquitination of Rev stabilizes the viral protein but hinders its activity.

Structural Basis of Ubiquitin Recognition by the Deubiquitinating Protease USP2

SummaryDeubiquitinating proteases reverse protein ubiquitination and rescue their target proteins from destruction by the proteasome. USP2, a cysteine protease and a member of the ubiquitin specific protease family, is overexpressed in prostate cancer and stabilizes fatty acid synthase, which has been associated with the malignancy of some aggressive prostate cancers. Here, we report the structure of the human USP2 catalytic domain in complex with ubiquitin. Ubiquitin uses two major sites for the interaction with the protease. Both sites are required simultaneously, as shown by USP2 inhibition assays with peptides and ubiquitin mutants. In addition, a layer of ordered water molecules mediates key interactions between ubiquitin and USP2. As several of those molecules are found at identical positions in the previously solved USP7/ubiquitin-aldehyde complex structure, we suggest a general mechanism of water-mediated ubiquitin recognition by USPs.

Quantitative analysis of in vitro ubiquitinated cyclin B1 reveals complex chain topology

Protein ubiquitination regulates many cellular processes, including protein degradation, signal transduction, DNA repair and cell division. In the classical model, a uniform polyubiquitin chain that is linked through Lys 48 is required for recognition and degradation by the 26S proteasome. Here, we used a reconstituted system and quantitative mass spectrometry to demonstrate that cyclin B1 is modified by ubiquitin chains of complex topology, rather than by homogeneous Lys 48-linked chains. The anaphase-promoting complex was found to attach monoubiquitin to multiple lysine residues on cyclin B1, followed by poly-ubiquitin chain extensions linked through multiple lysine residues of ubiquitin (Lys 63, Lys 11 and Lys 48). These heterogeneous ubiquitin chains were sufficient for binding to ubiquitin receptors, as well as for degradation by the 26S proteasome, even when they were synthesized with mutant ubiquitin that lacked Lys 48. Together, our observations expand the context of what can be considered to be a sufficient degradation signal and provide unique insights into the mechanisms of substrate ubiquitination.

Population of On-pathway Intermediates in the Folding of Ubiquitin

The role that intermediate states play in protein folding is the subject of intense investigation and in the case of ubiquitin has been controversial. We present fluorescence-detected kinetic data derived from single and double mixing stopped-flow experiments to show that the F45W mutant of ubiquitin (WT*), a well-studied single-domain protein and most recently regarded as a simple two-state system, folds via on-pathway intermediates. To account for the discrepancy we observe between equilibrium and kinetic stabilities and m-values, we show that the polypeptide chain undergoes rapid collapse to an intermediate whose presence we infer from a fast lag phase in interrupted refolding experiments. Double-jump kinetic experiments identify two direct folding phases that are not associated with slow isomerisation reactions in the unfolded state. These two phases are explained by kinetic partitioning which allows molecules to reach the native state from the collapsed state via two possible competing routes, which we further examine using two destabilised ubiquitin mutants. Interrupted refolding experiments allow us to observe the formation and decay of an intermediate along one of these pathways. A plausible model for the folding pathway of ubiquitin is presented that demonstrates that obligatory intermediates and/or chain collapse are important events in restricting the conformational search for the native state of ubiquitin.

Folding of Ubiquitin: A Simple Model Describes the Strange Kinetics

The ubiquitin mutant UbG folding experiments of Sabelko et al., in which "strange kinetics" were observed, are interpreted in terms of a simple kinetic model. A minimal set of states consisting of a semicompact globule, two off-pathway traps, and the native state are included; the fully unfolded state is not considered because folding to the semicompact globule is fast. Both the low- and the high-temperature experiments of Sabelko et al. are fitted by a system of kinetic equations determining the transitions between these states. It is possible that cold- and heat-denaturated states of UbG are the basis of the off-pathway traps. The fits of the kinetic model to the experimental results provides an estimate of the rate constants for the various reaction channels and show how their contributions vary with temperature. Introduction of an on-pathway intermediate instead of one of the off-pathway traps does not lead to agreement with the experiments.

Structures of Ubiquitin Insertion Mutants Support Site-specific Reflex Response to Insertions Hypothesis

We previously concluded that, judging from NMR chemical shifts, the effects of insertions into ubiquitin on its conformation appear to depend primarily on the site of insertion rather than the sequence of the insertion. To obtain a more complete and atomic-resolution understanding of how these insertions modulate the conformation of ubiquitin, we have solved the crystal structures of four insertional mutants of ubiquitin. Insertions between residues 9 and 10 of ubiquitin are minimally perturbing to the remainder of the protein, while larger alterations occur when the insertion is between residues 35 and 36. Further, the alterations in response to insertions are very similar for each mutant at a given site. Two insertions, one at each site, were designed from structurally homologous proteins. Interestingly, the secondary structure within these five to seven amino acid residue insertions is conserved in the new protein. Overall, the crystal structures support the previous conclusion that the conformational effects of these insertions are determined largely by the site of insertion and only secondarily by the sequence of the insert.

Differential Modification of Ras Proteins by Ubiquitination

Ras proteins are essential components of signal transduction pathways that control cell proliferation, differentiation, and survival. It is well recognized that the functional versatility of Ras proteins is accomplished through their differential compartmentalization, but the mechanisms that control their spatial segregation are not fully understood. Here we show that HRas is subject to ubiquitin conjugation, whereas KRas is refractory to this modification. The membrane-anchoring domain of HRas is necessary and sufficient to direct the mono- and diubiquitination of HRas. Ubiquitin attachment to HRas stabilizes its association with endosomes and modulates its ability to activate the Raf/MAPK signaling pathway. Therefore, differential ubiquitination of Ras proteins may control their location-specific signaling activities.

RNA Polymerase II Blockage by Cisplatin-damaged DNA: STABILITY AND POLYUBIQUITYLATION OF STALLED POLYMERASE

The consequences of human RNA polymerase II (pol II) arrest at the site of DNA damaged by cisplatin were studied in whole cells and cell extracts, with a particular focus on the stability of stalled pol II and its subsequent ubiquitylation. Site-specifically platinated DNA templates immobilized on a solid support were used to perform in vitro transcription in HeLa nuclear extracts. RNA elongation was completely blocked by a cisplatin intrastrand cross-link. The stalled polymerase was quite stable, remaining on the DNA template in nuclear extracts. The stability of pol II stalled at the site of cisplatin damage was also observed in live cells. A cell fractionation experiment using cisplatin-treated HeLa cells revealed an increased level of chromatin-associated pol II proteins following DNA damage. The stalled polymerase was transcriptionally active and capable of elongating the transcript following chemical removal of platinum from the template. Transcription inhibition by alpha-amanitin in vitro enhanced pol II ubiquitylation at ubiquitin residues Lys-6, Lys-48, and Lys-63. In live cells expressing epitope-tagged ubiquitin mutants, several ubiquitin lysines also participated in pol II ubiquitylation following DNA damage. Cisplatin treatment triggered ubiquitylation-mediated pol II degradation in HeLa cells, which could be prevented by the proteasomal inhibitor MG132. Fractionation of pol II from cells co-treated with MG132 and cisplatin indicated that the undegraded ubiquitylated polymerase was mostly unbound or only loosely associated with chromatin. These data are consistent with a model in which only a fraction of pol II, ubiquitylated in response to cisplatin damage of DNA, dissociates from the sites of platination. This altered polymerase is rapidly destroyed by proteasomes.

Ubiquitination of the Prototype Foamy Virus Envelope Glycoprotein Leader Peptide Regulates Subviral Particle Release

Foamy virus (FV) particle egress is unique among retroviruses because of its essential requirement for Gag and Env coexpression for budding and particle release. The FV glycoprotein undergoes a highly unusual biosynthesis resulting in the generation of three particle-associated, mature subunits, leader peptide (LP), surface (SU), and transmembrane (TM), derived from a precursor protein by posttranslational proteolysis mediated by furin or furinlike proteases. Previously at least three LP products of different molecular weights were detected in purified FV particles. Here we demonstrate that the higher-molecular-weight forms gp28LP and gp38LP are ubiquitinated variants of the major gp18LP cleavage product, which has a type II membrane topology. Furthermore, we show that all five lysine residues located within the N-terminal 60-amino-acid cytoplasmic domain of gp18LP can potentially be ubiquitinated, however, there seems to be a preference for using the first three. Inactivation of ubiquitination sites individually resulted in no obvious phenotype. However, simultaneous inactivation of the first three or all five ubiquitination sites in gp18LP led to a massive increase in subviral particles released by these mutant glycoproteins that were readily detectable by electron microscopy analysis upon expression of the ubiquitination-deficient glycoprotein by itself or in a proviral context. Surprisingly, only the quintuple ubiquitination mutant showed a two- to threefold increase in single-cycle infectivity assays, whereas all other mutants displayed infectivities similar to that of the wild type. Taken together, these data suggest that the balance between viral and subviral particle release of FVs is regulated by ubiquitination of the glycoprotein LP.

Lysosomal Degradation of CD33, the Target for the Anti-Leukemia Immunoconjugate, Gemtuzumab Ozogamicin: Evidence for Cbl-Mediated Monoubiquitination.

Background: CD33, the target for the anti-leukemia immunoconjugate, gemtuzumab ozogamicin (GO; Mylotarg™), is a transmembrane glycoprotein that contains two cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Although we have previously shown that disruption of the ITIMs prevents effective uptake of antibody-bound CD33 and significantly reduces GO-induced cytotoxicity, the mechanisms underlying this uptake and intracellular trafficking of the antibody-CD33 complex are not known. In this study, we tested whether CD33 is a target for monoubiquitination, a posttranslational modification that marks proteins for lysosomal degradation, and how this modification relates to tyrosine phosphorylation and endocytosis.Methods: CD33− Jurkat and 32D cells transduced with a lentiviral vector expressing either wild-type or mutant CD33 were analyzed for CD33 expression as well as internalization of anti-CD33 antibody by flow cytometry. Pull-down experiments were performed with glutathione S-transferase (GST) proteins fused to phosphorylated cytoplasmic tails of CD33 or TKB domain of Cbl/Cbl-b, using human myeloid cell lysates. For co-immunoprecipitation experiments, constructs encoding wild-type and mutant CD33, ubiquitin, Cbl/Cbl-b, and wild-type Fyn were transfected into HEK293T cells.Results: In engineered Jurkat cells, treatment with either anti-CD33 antibody or the tyrosine phosphatase inhibitor pervandate increased tyrosine phosphorylation of CD33. Pervanadate enhanced uptake of antibody-bound CD33; this effect was dependent upon the integrity of the ITIMs and was prevented by co-treatment with the Src tyrosine kinase inhibitor PP2. CD33 interacted with the TKB domains of Cbl and Cbl-b in GST fusion protein pulldown assays. Similarly, Cbl and Cbl-b were co-immunoprecipitated with CD33 in transfected 293T cells. Experiments in 293T cells further showed that CD33 is monoubiquitinated in an ITIM-dependent manner. Cbl or Cbl-b significantly increased the amount of CD33-associated ubiquitination; Cbl/Cbl-b-dependent ubiquitination could further be enhanced by co-transfected Fyn. Finally, 32D cells expressing lysine-to-arginine mutants of CD33 displayed much higher levels of surface CD33 but had reduced internalization of antibody-bound CD33 compared to cells expressing wild-type CD33, consistent with a reduced internalization/degradation of the lysine-to-arginine mutants.Conclusion: These studies indicate that Src-family kinase dependent phosphorylation favors internalization of antibody-bound CD33 and identify Cbl family proteins as potential binding partners of CD33. Importantly, CD33 is a target for ITIM-dependent monoubiquitination, and Cbl family proteins can act as an E3 ligase in this reaction. Our data therefore suggest a model where lysosomal routing and degradation of antibody-bound CD33 is secondary to Src-family kinase-induced phosphorylation of CD33 with subsequent phosphotyrosine-dependent binding of Cbl to and monoubiquitination of CD33.

Nuclear Dynamics of PCNA in DNA Replication and Repair

The DNA polymerase processivity factor proliferating cell nuclear antigen (PCNA) is central to both DNA replication and repair. The ring-shaped homotrimeric PCNA encircles and slides along double-stranded DNA, acting as a "sliding clamp" that localizes proteins to DNA. We determined the behavior of green fluorescent protein-tagged human PCNA (GFP-hPCNA) in living cells to analyze its different engagements in DNA replication and repair. Photobleaching and tracking of replication foci revealed a dynamic equilibrium between two kinetic pools of PCNA, i.e., bound to replication foci and as a free mobile fraction. To simultaneously monitor PCNA action in DNA replication and repair, we locally inflicted UV-induced DNA damage. A surprisingly longer residence time of PCNA at damaged areas than at replication foci was observed. Using DNA repair mutants, we showed that the initial recruitment of PCNA to damaged sites was dependent on nucleotide excision repair. Local accumulation of PCNA at damaged regions was observed during all cell cycle stages but temporarily disappeared during early S phase. The reappearance of PCNA accumulation in discrete foci at later stages of S phase likely reflects engagements of PCNA in distinct genome maintenance processes dealing with stalled replication forks, such as translesion synthesis (TLS). Using a ubiquitination mutant of GFP-hPCNA that is unable to participate in TLS, we noticed a significantly shorter residence time in damaged areas. Our results show that changes in the position of PCNA result from de novo assembly of freely mobile replication factors in the nucleoplasmic pool and indicate different binding affinities for PCNA in DNA replication and repair.

Lysine 63 Polyubiquitination of the Nerve Growth Factor Receptor TrkA Directs Internalization and Signaling

Nerve growth factor (NGF) binding to p75(NTR) influences TrkA signaling, yet the molecular mechanism is unknown. We observe that NGF stimulates TrkA polyubiquitination, which was attenuated in p75(-/-) mouse brain. TrkA is a substrate of tumor necrosis factor receptor-associated factor 6 (TRAF6), and expression of K63R mutant ubiquitin or an absence of TRAF6 abrogated TrkA polyubiquitination and internalization. NGF stimulated formation of a TrkA/p75(NTR) complex through the p62 scaffold, recruiting the E3/TRAF6 and E2/UbcH7. Peptide targeted to the TRAF6 binding site present in p62 blocked interaction with TRAF6 and inhibited ubiquitination of TrkA, signaling, internalization, and NGF-dependent neurite outgrowth. Mutation of K485 to R blocked TRAF6 and NGF-dependent polyubiquitination of TrkA, resulting in retention of the receptor on the membrane and an absence in activation of specific signaling pathways. These findings reveal that polyubiquitination serves as a common platform for the control of receptor internalization and signaling.

Ubiquitin Manipulation by an E2 Conjugating Enzyme Using a Novel Covalent Intermediate

Degradation of misfolded and damaged proteins by the 26 S proteasome requires the substrate to be tagged with a polyubiquitin chain. Assembly of polyubiquitin chains and subsequent substrate labeling potentially involves three enzymes, an E1, E2, and E3. E2 proteins are key enzymes and form a thioester intermediate through their catalytic cysteine with the C-terminal glycine (Gly76) of ubiquitin. This thioester intermediate is easily hydrolyzed in vitro and has eluded structural characterization. To overcome this, we have engineered a novel ubiquitin-E2 disulfide-linked complex by mutating Gly76 to Cys76 in ubiquitin. Reaction of Ubc1, an E2 from Saccharomyces cerevisiae, with this mutant ubiquitin resulted in an ubiquitin-E2 disulfide that could be purified and was stable for several weeks. Chemical shift perturbation analysis of the disulfide ubiquitin-Ubc1 complex by NMR spectroscopy reveals an ubiquitin-Ubc1 interface similar to that for the ubiquitin-E2 thioester. In addition to the typical E2 catalytic domain, Ubc1 contains an ubiquitin-associated (UBA) domain, and we have utilized NMR spectroscopy to demonstrate that in this disulfide complex the UBA domain is freely accessible to non-covalently bind a second molecule of ubiquitin. The ability of the Ubc1 to bind two ubiquitin molecules suggests that the UBA domain does not interact with the thioester-bound ubiquitin during polyubiquitin chain formation. Thus, construction of this novel ubiquitin-E2 disulfide provides a method to characterize structurally the first step in polyubiquitin chain assembly by Ubc1 and its related class II enzymes.

Site-specific Reflex Response of Ubiquitin to Loop Insertions

Predicting the structural effects of insertions in proteins by homology modeling remains a challenge. To investigate the molecular basis for conformational adaptations to insertions, ten mutants of ubiquitin were generated by introducing five different inserts, varying from five to 11 residues in size, at two different sites. Most insertion sequences were derived from homologous positions in structurally homologous ubiquitin-like proteins; to test sequence specificity, insertions were made into both homologous and non-homologous sites in ubiquitin. Structural inferences from NMR data suggest that each insertion site shows a reflex response to insertions: the sequence of the insertion has much less impact on structural adaptations than does the site of the insertion. Further, each site responds to insertions in a unique but consistent manner. For a given insertion site, different inserted sequences give rise to different stabilities, but the relationship between stability and sequence is not yet clear. However, the change in stability is similar for all insertions in a given site.

Ubiquitin-dependent Degradation of Interferon Regulatory Factor-8 Mediated by Cbl Down-regulates Interleukin-12 Expression

Interferon regulatory factor (IRF)-8/interferon consensus sequence-binding protein is regulated by both transcription and degradation. IRF-8 induced in peritoneal macrophages by interferon-gamma and lipopolysaccharide was degraded rapidly, and degradation of IRF-8 was blocked by MG132, the proteasome inhibitor, but inhibitors of calpain and lysosomal enzymes had no effect. The ubiquitination of IRF-8 was shown by co-immunoprecipitation from RAW264.7 macrophages retrovirally transduced with IRF-8 and hemagglutinin-ubiquitin. The dominant negative ubiquitin mutants K48R and K29R inhibited IRF-8 degradation in 293T cells, confirming the relationship between ubiquitination of IRF-8 and its degradation. IRF-8 carboxyl-terminal truncation mutants were not ubiquitinated and were consequently stable, indicating that the carboxyl-terminal domain of IRF-8 controls ubiquitination. The ubiquitin-protein isopeptide ligase (E3) that ubiquitinated IRF-8 was likely to be Cbl, which formed a complex with IRF-8, demonstrable by both immunoprecipitation and gel filtration. Furthermore, IRF-8 stability was increased by dominant negative Cbl, and IRF-8 ubiquitination was significantly attenuated in Cbl-/- cells. Reflecting increased stability and expression, the IRF-8 carboxyl-terminal deletion mutant induced interleukin (IL)-12 p40 promoter activity much more strongly than IRF-8 did. Furthermore, IRF-8-induced IL-12 p40 synthesis in RAW264.7 cells was enhanced by dominant negative Cbl, and peritoneal macrophages from Cbl-/- mice showed increased IL-12 p40 protein production. Taken together, these results suggest that the proteasomal degradation of IRF-8 mediated by the ubiquitin E3 ligase Cbl down-regulates IL-12 expression.

Lys6-modified Ubiquitin Inhibits Ubiquitin-dependent Protein Degradation

Ubiquitin plays essential roles in various cellular processes; therefore, it is of keen interest to study the structure-function relationship of ubiquitin itself. We investigated the modification of Lys(6) of ubiquitin and its physiological consequences. Mass spectrometry-based peptide mapping and N-terminal sequencing demonstrated that, of the 7 Lys residues in ubiquitin, Lys(6) was the most readily labeled with sulfosuccinimidobiotin. Lys(6)-biotinylated ubiquitin was incorporated into high molecular mass ubiquitin conjugates as efficiently as unmodified ubiquitin. However, Lys(6)-biotinylated ubiquitin inhibited ubiquitin-dependent proteolysis, as conjugates formed with Lys(6)-biotinylated ubiquitin were resistant to proteasomal degradation. Ubiquitins with a mutation of Lys(6) had similar phenotypes as Lys(6)-biotinylated ubiquitin. Lys(6) mutant ubiquitins (K6A, K6R, and K6W) also inhibited ATP-dependent proteolysis and caused accumulation of ubiquitin conjugates. Conjugates formed with K6W mutant ubiquitin were also resistant to proteasomal degradation. The dominant-negative effect of Lys(6)-modified ubiquitin was further demonstrated in intact cells. Overexpression of K6W mutant ubiquitin resulted in accumulation of intracellular ubiquitin conjugates, stabilization of typical substrates for ubiquitin-dependent proteolysis, and enhanced susceptibility to oxidative stress. Taken together, these results show that Lys(6)-modified ubiquitin is a potent and specific inhibitor of ubiquitin-mediated protein degradation.

Elevation of the Hsp70 chaperone does not effect toxicity in mouse models of familial amyotrophic lateral sclerosis

Mutations in copper/zinc superoxide dismutase (SOD1) account for 10-20% of a familial form of amyotrophic lateral sclerosis (ALS). A common feature of SOD1 mutants is abnormal aggregation of the aberrant SOD1 in neurons and glia. We now report that in ALS transgenic mouse models the constitutively expressed heat shock protein 70 (Hsp70) is mislocalized into aggregates together with mutant SOD1 and ubiquitin. Forcing increased synthesis of Hsp70 ameliorates both aggregate formation and toxicity in primary motor neurons in culture. However, chronic increase in an inducible form of Hsp70 to about 10-fold its normal level is shown here not to affect disease course or pathology developed in mice from accumulation of any of three familial ALS causing SOD1 mutants with different underlying biochemical characteristics. Therefore, increasing Hsp70 to a level that is protective in mouse models of acute ischemic insult and selected neurodegenerative disorders is not sufficient to ameliorate mutant SOD1-mediated toxicity.

Extending the Folding Nucleus of Ubiquitin with an Independently Folding β-Hairpin Finger: Hurdles to Rapid Folding Arising from the Stabilisation of Local Interactions

The N-terminal beta-hairpin sequence of ubiquitin has been implicated as a folding nucleation site. To extend and stabilise the ubiquitin folding nucleus, we have inserted an autonomously folding 14-residue peptide sequence beta4 which in isolation forms a highly populated beta-hairpin (>70%) stabilised by local interactions. NMR structural analysis of the ubiquitin mutant (Ubeta4) shows that the hairpin finger is fully structured and stabilises ubiquitin by approximately 8kJmol(-1). Protein engineering and kinetic (phi(F)-value) analysis of a series of Ubeta4 mutants shows that the hairpin extension of Ubeta4 is also significantly populated in the transition state (phi(F)-values >0.7) and has the effect of templating the formation of native contacts in the folding nucleus of ubiquitin. However, at low denaturant concentrations the chevron plot of Ubeta4 shows a small deviation from linearity (roll-over effect), indicative of the population of a compact collapsed state, which appears to arise from over-stabilisation of local interactions. Destabilising mutations within the native hairpin sequence and within the engineered hairpin extension, but not elsewhere, eliminate this non-linearity and restore apparent two-state behaviour. The pitfall to stabilising local interactions is to present hurdles to the rapid and efficient folding of small proteins down a smooth folding funnel by trapping partially folded or misfolded states that must unfold or rearrange before refolding.